Processing of Peptides and Proteins

ABSTRACT

The invention provides novel methionine aminopeptidase enzymes and their use.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 12/471,884, filed May 26, 2009, which is acontinuation of U.S. patent application Ser. No. 12/177,539, filed Jul.22, 2008, which is a continuation of Ser. No. 11/424,870, filed Jun. 18,2006, which is a continuation of International ApplicationPCT/DK2004/000891, filed Dec. 20, 2004, which claims the benefit of U.S.Provisional Patent Application Nos. 60/531,790, filed Dec. 22, 2003 and60/539,290, filed Jan. 26, 2004 and Danish Patent Application No. PA2003 01892, filed Dec. 19, 2003.

FIELD OF THE INVENTION

The invention relates to a method for processing initiator methioninecontaining proteins by the enzyme Methionine Aminopeptidase and mutantsthereof to yield initiator methionine free peptides.

BACKGROUND OF THE INVENTION

Production of peptides by recombinant techniques using eitherprokaryotic or eukaryotic expression systems inherently yields thepeptide with a leading methionine amino acid. This amino acid may not bepresent in the native protein i.e. the variant of the peptide processedfor translocation. Obtaining the peptide without the leading methioninethus requires a further processing step. In the present invention thestep is performed by the enzyme Methionine Aminopeptidase, whichselectively cleaves the initiator methionine from the peptide.

Methionine Aminopeptidases (Met-AP's) are known in the art as enzymeswhich cleaves leading methionines, if the leading peptide sequence is ofa certain predetermined character. Wild-type Escherichia coli Met-APselectively cleaves after an initiator Met residue if the residue in theP1′ position is Gly, Ala, Ser, Thr, Pro, Val or Cys.

In the present invention the methionine aminopeptidases are improved byintroducing mutations in the substrate binding sites which results inmethionine aminopeptidases which cleaves the methionine regardless ofthe leading peptide sequence (P1′ position)

SUMMARY OF THE INVENTION

The invention provides novel mutant methionine aminopeptidases.

The invention provides isolated DNA encoding such methionineaminopeptidases.

The invention provides host cells for producing such methionineaminopeptidases.

The invention provides the use of the mutant methionine aminopeptidasefor processing of peptides with an initiator methionine amino acid intoa methionine free peptide.

The invention also provides the processing of specific peptides bymutant methionine aminopeptidases.

The invention also provides a method for separating the methioninecontaining starting material from the final cleaved product.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Example of a E. coli Met-AP mutant expression construct layout.Purification indicates tag for purification purposes. Protease indicatesprotease cleavage site.

FIG. 2: Expression in e.g. E. coli of (A) NT1-Enterokinase-Met-AP Y168Aor (B) NT1-Enterokinase-Met-AP Y168G, M206N, Q233A as indicated.

FIG. 3: hexa-His-Xa-Met-AP (M206A, Q233A) cleavage of Met-hIL-21

FIG. 4: Maldi-tof es mass spectrum of purified hexa-His-Xa-Met-AP M206A,Q233A.

FIG. 5: Purification chromotogram of the separation of the threecompounds Met-IL-21, IL-21 and pyroglutamine IL-21.

DEFINITIONS

P1 defines the first amino acid N-terminal to the recognition site forthe enzyme. P1′ denotes the amino acid adjacent to P1 towards theC-terminal. P1 in the present invention is methionine.

In the present invention substrate specificity means selectivity towardsthe P1′ position—which is the position just C-terminal to methionine.Wild-type Escherichia coli Met-AP exhibits the substrate specificity,that it selectively cleaves after an initiator Met residue if theresidue in the P1′ position is Gly, Ala, Ser, Thr, Pro, Val or Cys. Themutants of the present invention showing an extended substratespecificity means that further aminoacid can occupy the P1′ position andstill cleavage of the methionine is observed.

In the context of the present invention variant means a sequence whichhas maintained the qualitative activity of the parent sequence, ie asmethionine aminopeptidase, but wherein the sequence differs from theparent sequence by deletions, insertions, extension or substitution ofone or more amino acids of the parent sequence. Variants in principlealso includes fragments of any length provided the activity ismaintained.

In the context of the present invention chemical derivatives of aspecific protein means a derivative of the native protein which is not avariant, and which maintains the qualitative activity of the parentprotein sequence. The chemical derivative includes derivatives such asPEG-groups.

The terms peptide and proteins are used interchangeable and is not meantas indications or limitations as to size or function of the sequences.

DESCRIPTION OF THE INVENTION

The Met-Ap from E. coli has a substrate defining pocket (as part ofactive site) essentially, but probably not exclusively, defined by theamino acids Tyr 168, Met 206 and Gln 233. Mutating these positionsextends the enzymes substrate specificity. The novel E. coliaminopeptidases described in the present invention extends theapplicability of the Methionine aminopeptidases to be useful forremoving the initiating methionine from almost any type of protein orpeptide regardless of the amino acid sequence downstream of themethionine (P1′ position). Hence, the initiator methionine can beremoved from all initiator methionine containing peptides or proteins toproduce initiator methionine-free peptides or proteins.

The E. coli Methionine aminopeptidase gene was cloned and mutantversions have been created using site directed mutagenesis.

The mutants were expressed in E. coli and the resulting enzymes werepurified by conventional His-tag system. The enzyme can also be taggedby for example the FLAG-system or tagged and purified by othertechnologies as described in WO 03042249. Catalytic activity wasmonitored using initiator Met containing hIL-21 as a substrate.

In principle, the invention is generally applicable to any peptide. Theinvention is demonstrated as being useful for cleavage of the initiatormethionine for peptides such as hIL-21. hIL-21 is a model system for P1′position being a Gln. IL-21 is described in WO00/53761 and is describedas being effective in the treatment of cancer and viral infection amongothers. IL-20 is described in WO9927103. hGH refers to human GrowthHormone. Both are model systems for other aminoacids in P1′ position.

In an aspect the invention provides E. coli aminopeptidease variantswhich are mutated in the active site having extended substratespecificity the P1′ position relative to the wild type.

In an aspect the invention provides the E. coli methionineaminopeptidase variants as described above which extend the substratespecificity in P1′ position to include Asn, Leu, Ile, Phe, His, Gln orTrp as well as the aminoacids allowed in position P1′ by the wild type.In an aspect the invention provides E. coli methionine aminopeptidasesas described above, wherein the residues in positions 168, 206 or 233has been amended into a sequence different from Y168 and/or M206 and/orQ233.In an aspect the invention provides E. coli methionine aminopeptidasesas described above, comprising amendments of the amino acid in position168.In an aspect the invention provides E. coli methionine aminopeptidasesas described above comprising amendment in position 206.In an aspect the invention provides E. coli methionine aminopeptidasesas described above comprising amendment in position 233.In an aspect the invention provides E. coli methionine aminopeptidasesas described above, comprising amendments in position 206 and 233.In an aspect the invention provides E. coli methionine aminopeptidasesas described above comprising amendments in position 168 and 206.In an aspect the invention provides E. coli methionine aminopeptidasesas described above comprising amendments in position 168 and 233.In an aspect the invention provides E. coli methionine aminopeptidase asdescribed above, comprising amendments in positions 168, 206 and 233.In an aspect the invention provides E. coli methionine aminopeptidasesas described above, wherein the amendments comprises exchange ofwildtype amino acid into Gly, Ala, Ser, Thr, Asn or Asp.In an aspect the invention provides E. coli methionine aminopeptidasesas described above, wherein the amendments comprises Ala and/or Gly.In an aspect the invention provides E. coli methionine aminopeptidasesas described above, wherein the amendments comprises Ala.In an aspect the invention provides E. coli methionine aminopeptidase asdescribed above, wherein position 168 is Ala.In an aspect the invention provides E. coli methionine aminopeptidase asdescribed above, wherein position 206 is Ala.In an aspect the invention provides. E. coli methionine aminopeptidaseas described above, wherein position 233 is Ala.

The invention thus provides the methionine aminopeptidase enzyme havingthe following sequence (also described as seq. id. no. 1)

MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREAIGAIQKFVEAEGFSVVREXa CGHGIGRGFHEEPQVLHYDSRETNVVLKPGMTFTIEPXb VNAGKKEIRTMKDGWTVKTKDRSLSAXcYEHTIVVTDNGCEILTLRKDDTIPAIISHDE,

wherein X_(a), X_(b) and X_(c) are variable amino acids, and whereinX_(a), X_(b) and X_(c) are not simultaneously Tyr, Met and Glnrespectively. In an aspect of the invention one or more of X_(a), X_(b)and X_(c) are exchanged from the wild type amino acid into Gly, Ala,Ser, Thr, Asn or Asp. In an aspect of the invention X_(a), X_(b) andX_(c) are exchanged from the wild type amino acid into Gly or Ala. In anaspect of the invention X_(a), X_(b) and X_(c) are exchanged from thewild type amino acid into Ala. The present invention thus providessubstitution Y168 to Ala (Y168A) (Seq. id no. 9)

MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREACGHGIGRGFHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVT DNGCEILTLRKDDTIPAIISHDEand the corresponding DNA encoding the above as seq. id. no. 8;

The present invention thus provides substitution Met 206 to Ala (M206A)(Seq. id no. 3)

MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREXCGHGIGRGFHEEPQVLHYDSRETNVVLKPGMTFTIEPAVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILTLRKDDTIPAIISHDE,which extends the enzymes substrate specificity to allow the followingamino acids: Asn, Leu, Ile and Phe in the P1′ position.The present invention also provides substituting Gln 233 to Ala (Q233A)(Seq. id. No. 5)

MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREXCGHGIGRGFHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAAYEHTIVVTDNGCEILTLRKDDTIPAIISHDE,

or both Met 206 and Gln 233 into Ala (M206A Q233A) (Seq. id no. 7):

MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAV-SACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIV-GKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREXCGHGIGRG-FHEEPQVLHYDSRETNVVLKPGMTFTIEPAVNAGKKEIRTMKDGWTVKTKDRSLSAAYEHTIVVTDNGCEILTLRKDDTIPAIISHDE,which further allow the P1′ amino acids to be His, Gln and Trp.In aspects of the invention position 168 is amended into Gly (Y168G) orAla (Y168A) or Asn (Y168N). Aspects of the invention are wherein aminoacid 206 is an Ala (M206A) or a Gly (M206G) or Asn (M206N), and/orwherein amino acid 233 is an Ala (Q233A) or a Gly (Q233G) or Asn(Q233N). Aspects of the invention comprise the combination of two orthree amendments according to the below, —wherein the wild-typecombination of (Y168 M206 233Q) is not within the invention.

Position 168 Position 206 Position 233 A/G/N/Y A/G/N/M A/G/N/QAccordingly, aspects of the invention are wherein position 206 andposition 233 are both Ala (M206A Q233A) or Gly or Asn, or combinationsthereof: (M206G Q233A), (M206G Q233G), (M206A Q233G), (M206N Q233A),(M206N Q233N), (M206A Q233N). Aspects of the invention are whereinposition 168 is amended according to the below:

Position 168 Position 206 Position 233 A/G/N A Q A/G/N G Q A/G/N N QA/G/N M A A/G/N M G A/G/N M N A/G/N A A A/G/N G A A/G/N N A A/G/N A GA/G/N A N A/G/N N G A/G/N N N A/G/N G G A/G/N G NAspects of the invention are wherein at least one of the amendedpositions are amended into an Ala.Aspects of the invention are the following mutants: (Y168G M206A),(Y168G M206A 233A), (Y168G M206N), (Y168G M206N 233A), (Y168A M206A233A), (Y168A M206A), (Y168A M206N), (Y168A M206N 233A) and (M206AQ233A);

The invention thus provides a novel enzymes capable of cleaving apeptide containing an initiating methionine followed by a Asn, Leu, Ile,Phe, His, Gln or Trp in the P1′ position as well as the amino acidsallowed by the wild type E. coli aminopeptidase. Wildtype E. colimethionine aminopeptidase allows the P1′ to be any of the followingamino acids: Gly, Ala, Ser, Thr, Pro, Val or Cys. The invention thusalso provides recombinant DNA molecules encoding the sequence above. TheDNA sequences are disclosed in Seq. id. no. 2, 4 and 6. The inventionalso provides specifically the DNA encoding the sequences for themutants M206A, Q233A or M206A Q233A) above.

In the present invention the mutant Methionine aminopeptidases areexpressed in E. coli, but in principle the host cells could be of otherprokaryotic origin or eukaryotic origin such as Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoris etc. or forexample mammalian cells.The invention thus provides host cells transformed by the recombinantDNA molecule of above.Removal of initiator methionine by methionine aminopeptidase may beperformed in vitro following methionine aminopeptidase expression in,and purification from, prokaryotic or eukaryotic cells. This procedureis demonstrated below. Alternatively removal of initiator methionine maytake place in vivo either in cells expressing a di-cistronic plasmid orin cells co-expressing plasmids carrying the methionine aminopeptidaseand the substrate peptide or protein. In vivo initiator methionineprocessing may also be performed in cells where the genes encoding themethionine aminopeptidase and the peptide or protein to be processedhave been integrated into the genome.

Experiments have been performed which provides a set of optimumconditions for the reaction: The optimal temperature for the reactionwas determined to be between 15 and 24 degrees Celsius. Typically thereaction was hereafter performed at 18 degrees Celsius.

The concentration of ZnCl₂ was determined to be optimal at around 7.5 μMand NaCl concentration was found optimal around 100 mM and acceptableunder 130 mM.

After cleavage of the initiator methionine separation of the productfrom the starting material can be achieved by exploiting the differentbiophysical properties of the two peptides.

In an embodiment of the invention the peptide is hIL-21, which afterremoval of the initial methionine contains a Gln in the N-terminal.Treatment with Qcyclase forms a pyroglutamine (pGlu). Due to cyclic amidformation the products net change is negative relative to the Methioninecontaining peptide. The difference in charge affects the eluation on acation exchange column, due to Methionine containing peptide having astronger binding to the cation resin. Further, in non-cyclised hIL-21the N-terminal positioned Gln residue will have the ability to form ahydrogen bond between the side chain amide oxygen and the chargedN-terminal backbone amine and thereby masking the charge at theN-terminus. Met-hIL-21 will not possess the ability for a similar chargemasking and will therefore bind stronger to the cation exchange columnthan hIL-21.

In an embodiment of the invention, a method separation of proteinmixtures between identical proteins starting with Met-Gln and Glnrespectively is provided.

In a specific embodiment of the invention separation of Met-hIL-21 andhIL-21 is provided.

In another specific embodiment of the invention separation of Met-hIL-21and hIL-21 and mutants thereof is provided.

EXAMPLES

Various Met-AP expression constructs, as outlined in FIG. 1, have beencreated. NT1 (HHHNSWDHDINR) or hexa-His tag has been added to thevarious mutant forms of Met-AP for purification purposes. Thepurification tag may be removed using Factor Xa in some constructs orEnterokinase in others, or the purification tag may be left on theenzyme. mRNA expression was under the control of the T7 or the tacpromoter. Constructs under the control of the T7 promoter were expressedin BL21(DE3) whereas constructs under the control of the tac promoterwere expressed in BL21. Expression was induced by addition of IPTG to0.4 mM to cultures (6 mL) grown to OD₆₀₀ 0.4 in LB-medium. Cells wereharvested by centrifugation after 2.5 hours. Cell lysis was done bymultiple freeze-thaw cycles and soluble or insoluble protein fractionswere separated by centrifugation. Soluble or insoluble protein, beforeor after induction of expression, originating from equal amounts cells(measured by OD₆₀₀) were subjected to SDS-PAGE and subsequent colloidalblue staining (FIG. 2). Met-AP expression levels were estimated at ˜250mg/L after 2.5 h of induction in 6 mL cultures.

E. coli cells harvested from 1 L of culture expressing hexa-His-Met-APM206A, Q233A were lysed using a cell disruptor, and the clarified lysatewas applied on a Ni²⁺-NTA superflow column. Elution with an imidazolegradient released the Met-AP fusion protein at approximately 200 mMimidazole. The enzyme was further purified and buffer exchanged (intostorage/cleavage buffer) using size exclusion chromatography. The enzymewas analysed using SDS-PAGE, MALDI-MS and N-terminussequencing—verifying the molecular mass and identity of the enzyme.

According to the procedure above NT1-Enterokinase-Met-AP mutants wereprepared. Expression was under the control of the tac promoter. Additionof IPTG to the cultures induced primarily soluble expression of theMet-AP enzymes. The following mutants were prepared according to theabove: (Y168G M206A), (Y168G M206A 233A), (Y168G M206N), (Y168G M206N233A), (Y168A M206A 233A), (Y168A M206A), (Y168A M206N) and (Y168A M206N233A)

Hexa-His-Xa-MetAP Q233A was affinity purified using Ni²⁺-NTA superflow.Maldi-tof es mass spectrum of purified hexa-His-Xa-Met-AP M206A, Q233Ashows that the correct enzymes were isolated. A mass of 32038.90corresponds to Met-hexa-His-Xa-Met-AP M206A, Q233A and a mass of31942.10 corresponds to hexa-His-Xa-Met-AP M206A, Q233A indicating thathexa-His-Xa-Met-AP M206A, Q233A was processed by WT Met-AP orhexa-His-Xa-Met-AP M206A, Q233A in vivo. The result is demonstrated inFIG. 4.

Addition of hexa-His-Xa-Met-AP M206A, Q233A to Met-hIL-21 at pH 7, 18°C. generated ˜65% Met-free hIL-21. In 44 h. more than 90% cleavage ofMet-hIL-21 could be observed (FIG. 3).

Another mutant prepared by this method was hexa-His-Xa-Met-AP Q233A;

Removal of Initiator Met from Met-IL21 by Met-AP (M206A, Q233A).

Purified Met-AP (M206A, Q233A) was used to remove the initiatorMethionine from partly or fully purified Met-IL21. The cleavage wasperformed in a reaction buffer typically consisting of the followingcomponents: 2-100 mM K₂SO₄, 2-500 mM NaCl, 1-100 μM ZnCl₂ and 2-30 mMHepes buffer pH 6-8. The cleavage was assayed by MALDI-TOF spectroscopy.The time of reaction was 2-66 hours. Using these condition removal ofMethionine from Met-IL21 below detection limits of Met-IL21 could beperformed.

Removal of Initiator Met from Met-IL21 by Met-AP (M206A).

Purified Met-AP (M206A) was used to remove the initiator Methionine frompartly or fully purified Met-IL21. The cleavage was performed in areaction buffer typically consisting of the following components: 2-100mM K₂SO₄, 2-500 mM NaCl, 1-100 μM ZnCl₂ and 2-30 mM Hepes buffer pH 6-8.The cleavage was assayed by MALDI-TOF spectroscopy. The time of reactionwas 2-66 hours. Using these condition removal of Methionine fromMet-IL21 below detection limits of Met-IL21 could be performed.

Influence of Temperature on Removal of Initiator Met from Met-IL21 byMet-AP (M206A, Q233A).

Using the conditions and assay described in example 1 the temperaturewas varied between 4, 15, 24 and 30 degrees Celsius, respectively whilethe other parameters was fixed. The optimal temperature for the reactionwas determined to be between 15 and 24 degrees Celsius. Typically thereaction was hereafter performed at 18 degrees Celsius.

Influence of ZnCl₂ Concentration on Removal of Initiator Met fromMet-IL21 by Met-AP (M206A, Q233A).

Using the conditions and assay described in example 1 the ZnCl₂concentration was varied between 7.5, 11 and 15 μM, respectively whilethe other parameters were fixed. The optimal ZnCl₂ concentration for thereaction was determined to be 7.5 μM. Typically, the reaction washereafter performed at 7.5 μM ZnCl₂.

Influence of NaCl Concentration on Removal of Initiator Met fromMet-IL21 by Met-AP (M206A, Q233A).

Using the conditions and assay described in example 1 the NaClconcentration was varied between 80, 130 and 180 mM, respectively whilethe other parameters were fixed. The maximum NaCl concentrationtolerated for the reaction to run was determined to be 130 mM. Typicallythe reaction was hereafter performed at 100 mM NaCl.

Influence of the Addition of Q-cyclase on Removal of Initiator Met fromMet-IL21 by Met-AP (M206A, Q233A) and the Formation of Pyroglutamine.

Using the conditions as described in examples above the effect of addingQ-cyclase to the reaction mixture was determined. Again MALDI-TOF wasused for assaying the removal of Methonine and subsequently conversionof glutamine in position 1 in IL21 into pyro-glutamine. It was foundthat the addition of Q-cyclase to the reaction mixture did notnegatively influence the removal of initiator Methionine from Met-IL21and further the Q-cyclase was fully efficient in converting glutamine inposition 1 in IL21 into pyro-glutamine under the reaction conditionsdescribed in the examples above.

Purification and Separation of Met-IL21, IL21 and Pyro-Glutamine IL-21Using a Mono-S Column.

The different bio-physical properties between Met-IL21, IL21 andpyro-glutamine IL21 can be used for purification purposes/separation.Pyro-glutamine IL21 will due to the cyclized amid formation lack thenormal protonation of the N-terminus. The (−1) charge difference betweenhIl-21 starting with pyro-glutamine and Met-IL21 can be used on a cationexchange column that will elute pyroglutamine IL21 first (due to itslack of one positive charge) and subsequently Met-IL21 which displays astronger binding to the cation resin. Further, in non-cyclized IL21 theN-terminally positioned glutamine will have the ability to form ahydrogen bond between the side chain amide oxygen and the chargedN-terminal backbone amine, and thereby masking the charge at theN-terminus. Met-IL21 will not poses the ability for a similar chargemasking and will therefore bind stronger to a cation exchange columnthan IL21. A mixture of Met-IL21, IL21 and pyro-glytamine IL21 including300 mM NaCl and buffered at pH 6.5 was loaded on a Mono-S column. The Abuffer consisted of 300 mM NaCl buffered at pH 6.5 and the B buffer 1 MNaCl buffered at pH 6.5. A linear gradient (performed on an AKTA system)from 0-20% B buffer was applied over 45 column volumes. The fractionswas assayed as described under the Q-cyclase example above. Using theabove described gradient, efficient separation of Met-IL21, IL21.pyro-glutamine IL21 was achieved. (FIG. 5)

Met-hGH

Purified Met-AP (M206A) is used to remove the initiator Methionine frompartly or fully purified Met-hGH (human growth hormone) where the P1′ isa Phe residue. The cleavage is performed in a reaction buffer typicallyconsisting of the following components: 2-100 mM K₂SO₄, 2-500 mM NaCl,1-100 μM ZnCl₂ and 2-30 mM Hepes buffer pH 6-8. The cleavage is assayedby MALDI-TOF spectroscopy. The time of reaction is 2-66 hours. Usingthese conditions partly or full removal of Methionine from Met-hGH isdemonstrated.

Met-hGH

Purified Met-AP (M206A, Q233A) is used to remove the initiatorMethionine from partly or fully purified Met-hGH (human growth hormone)where the P1′ is a Phe residue. The cleavage is performed in a reactionbuffer typically consisting of the following components: 2-100 mM K₂SO₄,2-500 mM NaCl, 1-100 μM ZnCl₂ and 2-30 mM Hepes buffer pH 6-8. Thecleavage is assayed by MALDI-TOF spectroscopy. The time of reaction is2-66 hours. Using these conditions partly or full removal of Methioninefrom Met-hGH is achieved.

Met-IL-20

Purified Met-AP (M206A) is used to remove the initiator Methionine frompartly or fully purified Met-IL-20 where the P1′ is a Leu residue. Thecleavage is performed in a reaction buffer typically consisting of thefollowing components: 2-100 mM K₂SO₄, 2-500 mM NaCl, 1-100 μM ZnCl₂ and2-30 mM Hepes buffer pH 6-8. The cleavage is assayed by MALDI-TOFspectroscopy. The time of reaction is 2-66 hours. Using these conditionspartly or full removal of Methionine from Met-IL-20 is demonstrated.

Met-IL-20

Purified Met-AP (M206A, Q233A) is used to remove the initiatorMethionine from partly or fully purified Met-IL-20 where the P1′ is aLeu residue. The cleavage is performed in a reaction buffer typicallyconsisting of the following components: 2-100 mM K₂SO₄, 2-500 μM NaCl,1-100 μM ZnCl₂ and 2-30 mM Hepes buffer pH 6-8. The cleavage is assayedby MALDI-TOF spectroscopy. The time of reaction is 2-66 hours. Usingthese conditions partly or full removal of Methionine from Met-IL-20 isdemonstrated.

1. An E. coli aminopeptidease variant comprising one or more amino acidsequence variations in the substrate defining pockets.
 2. Theaminopeptidase variant of claim 1, wherein the variant has extendedsubstrate specificity the P1′ position relative to the wild type E. coliaminopeptidase.
 3. The aminopeptidase variant of claim 2, wherein theaminopeptidase variant has extended substrate specificity in P1′position to include Asn, Leu, Ile, Phe, His, Gln or Trp as well as theamino acids allowed in position P1′ by the wild type.
 4. Theaminopeptidase variant of claim 3, wherein the residues in positions168, 206 and/or 233 (of the wild type aminopeptidase) are substituted.5. The aminopeptidase variant of claim 4, wherein the residue inposition 168 is substituted.
 6. The aminopeptidase variant of claim 4,wherein the residue in position 206 is substituted.
 7. Theaminopeptidase variant of claim 4, wherein the residue in position 233is substituted.
 8. The aminopeptidase variant of claim 4, wherein theresidue in positions 206 and 233 are substituted.
 9. The aminopeptidasevariant of claim 4, wherein the residue in positions 168 and 206 aresubstituted.
 10. The aminopeptidase variant of claim 4, wherein theresidue in positions 168 and 233 are substituted.
 11. The aminopeptidasevariant of claim 4, wherein the residue in positions 168, 206 and 233are substituted.
 12. The aminopeptidase variant of claim 4, wherein thevariant comprises one or more substitutions with amino acid residuesselected from the group consisting of Gly, Ala, Ser, Thr, Asn or Asp.13. The aminopeptidase variant of claim 12, wherein the substitution isselected from Gly and Ala.
 14. The aminopeptidase variant of claim 13,wherein the substitution is with an Ala residue.
 15. The aminopeptidasevariant of claim 14, wherein the variant comprises a substitution atposition 168 with an Ala residue.
 16. The aminopeptidase variant ofclaim 14, wherein the variant comprises a substitution at position 206with an Ala residue.
 17. The aminopeptidase variant of claim 14, whereinthe variant comprises a substitution at position 233 with an Alaresidue.
 18. The aminopeptidase variant of claim 16, wherein the variantcomprises substitutions at positions 206 and 233 with Ala.
 19. Theaminopeptidase variant of claim 1, wherein the variant comprises SEQ IDNO:
 3. 20. The aminopeptidase variant of claim 1, wherein the variantcomprises SEQ ID NO:
 5. 21. The aminopeptidase variant of claim 1,wherein the variant comprises SEQ ID NO:
 7. 22. The aminopeptidasevariant of claim 1, wherein the variant comprises SEQ ID NO:
 9. 23. Amethod of removing an initiator methionine from a peptide comprisingcontacting the peptide with an amino peptidase variant according toclaim 1 under conditions suitable for the aminopeptidase variant toremove the initiator methionine.
 24. The method of claim 23, wherein thepeptide is selected from (a) a recombinant interleukin-21 (IL-21); (b)an IL-21 variant; or (c) a derivative of either (a) or (b).
 25. Themethod of claim 23, wherein the peptide is recombinantinterleukin-2-(IL-20).
 26. The method of claim 23, wherein the peptideis recombinant human growth hormone (hGH).
 27. The method of claim 24,wherein the peptide is recombinant human IL-21.
 28. A method ofpurifying a peptide product having a Gln residue in the N-terminus froma first peptide comprising the Gln residue and an initiator Met residue,the method comprising applying Qcyclase to the first peptide andpurifying the peptide product obtained thereby based on chargedifferences.
 29. The method of claim 28, wherein the peptide product ispurified on a cation exchange column.